The present invention relates to nanosyringes, and in particular to a nanosyringe array and method of making the nanosyringe array.
Microinjection techniques have been used for a variety of applications, including high-efficiency transformation, protein injection, pathogen injection and organelle transfer. Transfer of DNA to mammalian cell cultures and embryos using microinjection has also been performed. More recently, microinjection has been employed to develop transgenic animals for pharmacological studies in the cardiovascular system, endocrine system, cancer and toxicology. It has also been used to examine the role of the c-fos gene as a mitogenic signal in mammalian cells by injection of protein inhibitors and monoclonal antibodies that block mitogenic activities.
Micropipettes are primarily constructed of tapered borosilicate glass, quartz, or aluminosilicate needles with a minimum diameter of between 50 and 100 nm. The primary disadvantages of these pipettes include inherent damage inflicted on host cells, the inability to accurately control injection rates, the inability to inject more than one cell at a time, and the inability to inject more than one sample into a given cell at one time. Recently, a galinstan expansion femtosyringe was formed that reduced the damage inflicted on host cells. In addition, heat induced galinstan (also known as gallinstan, a liquid metal alloy of gallium, indium and tin) was used to accurately control the rate of injection. These femtosyringes permit the injection of subcellular organelles such as vacuoles, mitochondria and chloroplasts while maintaining the integrity of cell membranes. Such femtosyringes are expensive to form, and do not facilitate the injection of more than one cell at a time, nor do they provide the ability to inject different substances simultaneously into the same cell. Expensive needle puller equipment is also required to form femtosyringes.
In one attempt to provide an array of microneedles, a plurality of parallel hollow non-silicon microneedles are formed on a planar surface of a substrate. Multiple arrays of these needles can be coupled to form a three dimensional array with the substrates still attached, or removed. Cross coupling channels provide for free fluid flow. The array is used to increase the flow rate of a fluid to be injected. Further, the size of the needles constructed using this technique are much larger than those required to permit the injection of subcellular organelles, and may lead to unacceptable damage to cellular structures.
A nanosyringe is constructed using micro and nano fabrication techniques on a substrate. In one embodiment, the nanosyringe is formed as a membrane of silicon carbide or silicon nitride on a silicon substrate using photolithography or other means. The nanosyringe comprises a tip for penetrating a host without destroying the integrity of a host membrane. As used herein, the term needle is interchangeable with the term syringe.
In one embodiment, an array of nanosyringes comprises a large number of independently controlled nanosyringes suitable for injecting a large number of cells or other structures at a given time, or injecting a variety of samples into a single cell at one time or at staggered time intervals. In one embodiment, each nanosyringe is independently controlled with respect to injection properties. The spacing of the nanosyringes is adjusted based upon a specific objective at the time of formation of the array. For example, arrays with a large spacing of 5-10 xcexcm may be used for injecting large numbers of cells. As another example, arrays with smaller spacing, such as less than 50 nm between tips, may be used for injection various samples into a single cell at specific rates, time intervals and location. They are further used to increase the flow rate of a sample to a cell. In one embodiment, a variety of samples can be injected in varying amounts and at varying times.
In one embodiment, the arrays are utilized to draw fluid or remove samples from cells. An external pumping system coupled to one or more nanosyringes allows non-destructive sampling of a cell matrix or organelles of a cell as well as real time sampling and analysis of physiological changes within an individual cell. In one embodiment, a nanosyringe both injects a first fluid and extracts a second fluid coincident with a single penetration of a host membrane.
In one embodiment, sensor and detection capabilities, as well as micro-pumps and valves are directly integrated into the system using micro and nano fabrication techniques on a semiconductor substrate. This provides the ability to instantaneously sample a cell""s cytoplasm following the addition of a particular drug injected into the nucleus of that cell. Arrays of nanosyringes are also formed for a variety of microfluidic systems where precise delivery of liquids is desired. In one embodiment, a system is provided to independently position individual nanosyringes within a three axis coordinate system.
In one embodiment, a silicon carbide nanosyringe is constructed using micro and nano fabrication techniques on a silicon substrate. Each nanosyringe is independently controlled with respect to injection properties. An external pump system coupled to a nanosyringe array allows non-destructive sampling of the cell""s matrix and organelles, and real time sampling and analysis of physiological changes within individual cells. Sensor and detection capabilities, as well as micro-pumps and valves are directly integrated into the system using micro and nano fabrication techniques on a semiconductor substrate.
The present subject matter includes fabrication of thin, suspended membranes supported by a silicon substrate. In various embodiments, the membrane includes thin film materials such as silicon nitride or silicon carbide. In one embodiment, the membrane is formed using a non-planar (that is, not flat) surface. The present subject matter includes membranes formed using a cylinder, column or cone.
Other aspects of the invention will be apparent on reading the following detailed description of the invention and viewing the drawings that form a part thereof.